The first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay. Clostridium botulinum type C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group III C. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibit C. botulinum toxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.

A total of 312 samples of sliced, vacuum packaged, cold-smoked pork from 15 meat processing plants in Latvia and Lithuania, obtained over a 15-month period from 2003 until 2004, were analyzed for the presence of Listeria monocytogenes at the end of their shelf-life. Overall, 120 samples (38%) tested positive for L. monocytogenes. Despite the long storing period, the levels of L. monocytogenes in cold-smoked pork products were low. Manufacturing processes were studied at seven meat processing plants. A new approach with a logistic multivariable regression model was applied to identify the main factors associated with L. monocytogenes contamination during the manufacturing of cold-smoked pork products. Brining by injection was a significant factor (odds ratio 10.66; P<0.05) for contamination of product with L. monocytogenes. Moreover, long cold-smoking times (>=12 h) had a significant predictive value (odds ratio 24.38; P<0.014) for a sample to test positive for L. monocytogenes. Pulsed-field gel electrophoresis results indicated that various sources of L. monocytogenes contamination existed over periods of time in several meat processing plants. In two meat processing plants, persistent L. monocytogenes strains belonging to serotypes 1/2a and 1/2c were found.

Persistent Listeria monocytogenes contamination of food industry equipment is a difficult problem to solve. Ultrasonic cleaning offers new possibilities for cleaning conveyors and other equipment that are not easy to clean. Ultrasonic cleaning was tested on three conveyor belt materials: polypropylene, acetal, and stainless steel (cold-rolled, AISI 304). Cleaning efficiency was tested at two temperatures (30 and 45 degrees C) and two cleaning times (30 and 60 s) with two cleaning detergents (KOH, and NaOH combined with KOH). Conveyor belt materials were soiled with milk-based soil and L. monocytogenes strains V1, V3, and B9, and then incubated for 72 h to attach bacteria to surfaces. Ultrasonic cleaning treatments reduced L. monocytogenes counts on stainless steel 4.61 to 5.90 log units; on acetal, 3.37 to 5.55 log units; and on polypropylene, 2.31 to 4.40 log units. The logarithmic reduction differences were statistically analyzed by analysis of variance using Statistical Package for the Social Sciences software. The logarithmic reduction was significantly greater in stainless steel than in plastic materials (P<0.001 for polypropylene, P=0.023 for acetal). Higher temperatures enhanced the cleaning efficiency in tested materials. No significant difference occurred between cleaning times. The logarithmic reduction was significantly higher (P=0.013) in cleaning treatments with potassium hydroxide detergent. In this study, ultrasonic cleaning was efficient for cleaning conveyor belt materials.

Contamination routes of Listeria monocytogenes were examined in a chilled food processing plant that produced ready-to-eat and ready-to-reheat meals during an 8-year period by amplified fragment length polymorphism (AFLP) analysis. A total of 319 L. monocytogenes isolates were recovered from raw materials (n=18), the environment (n=77), equipment (n=193), and products (n=31), and 18 different AFLP types were identified, five of which were repeatedly found to be persistent types. The three compartments (I to III) of the plant showed markedly different contamination statuses. Compartment I, which produced cooked meals, was heavily contaminated with three persistent AFLP types. AFLP type A1 dominated, and it comprised 93% of the isolates of the compartment. Compartment II, which produced uncooked chilled food, was contaminated with four persistent and five nonpersistent AFLP types. The equipment of compartment III, which produced cooked ready-to-reheat meals, was free of contamination. In compartments that produced cooked meals, the cleaning routines, product types, and lack of compartmentalization seemed to predispose production lines to persistent contamination. The most contaminated lines harbored L. monocytogenes in coolers, conveyors, and packing machines. Good compartmentalization limited the flow of L. monocytogenes into the postheat -treatment area and prevented the undesired movement of equipment and personnel, thus protecting the production lines from contamination. In compartment II, grated cheese was shown to cause product contamination. Therefore, special attention should be paid to continuous quality control of raw ingredients when uncooked ready-to-eat foods are produced. In compartment II, reconstruction of the production line resulted in reduced prevalence rates of L. monocytogenes and elimination of two persistent AFLP types.

The prevalences of various genotypes of enterotoxin gene-carrying (cpe-positive) Clostridium perfringens type A in 24 different food poisoning outbreaks were 75% (chromosomal IS1470-cpe), 21% (plasmid-borne IS1470-like-cpe), and 4% (plasmid-borne IS1151-cpe). The results show that C. perfringens type A carrying the plasmid-borne cpe is a common cause of food poisoning.

Glycogen debranching enzyme (GDE) is together with glycogen phosphorylase responsible for the degradation of glycogen. The present study compares the post-mortem activity of GDE and breakdown of the glycogen pools in M. longissimus dorsi of RN- carrier pigs and in wild type animals. The activity of GDE (n=14) and pH (n=20) was measured 0.5, 3, 5, 24 and 48 h post-mortem. The change in pro-glycogen and in macro-glycogen content (n=20) was followed until 216 h post-mortem and the transcription level of GDE, glycogenin and glycogen synthase m-RNA (n=19) were measured 0.5 h post-mortem. Both the activity of GDE and the transcription level of GDE were found to be similar in RN- carriers and wild type animals shortly after slaughter. However, the activity declined faster in wild type animals compared with RN- carriers with increasing time post-mortem. The contents of both pro-glycogen and macro-glycogen were higher in RN- carriers compared with wild type animals, and further, the proportion of macro-glycogen was higher in RN- carriers compared with wild type animals. During the post-mortem period, only degradation of pro-glycogen was observed in both genotypes. The decrease in pH was faster and the ultimate pH lower in RN- carriers than in wild type animals. It was suggested that the higher GDE activity in the late phase of the post-mortem period in muscles from RN- carriers renders the extended pH decrease in these muscles.

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (</=3%) in the nucleotide sequences of the gene cluster components were observed among the three strains and the published BoNT/E-producing clostridia. The orfx3, orfx2, orfx1, and p47 gene sequences of the three type E strains shared homologies of 81%, 67 to 76%, 78 to 79%, and 79 to 85%, respectively, with published sequences for type A1 and A2 C. botulinum. Analysis of bont/E from the 14 type E strains and 19 previously published BoNT/E-producing clostridia revealed six neurotoxin subtypes, with a new distinct subtype consisting of three Finnish isolates alone. The amino acid sequence of the subtype E6 neurotoxin differed 3 to 6% from the other subtypes, suggesting that these subtype E6 neurotoxins may possess specific antigenic or functional properties.

Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

The purpose of the study was to examine the activity of glycogen debranching enzyme, GDE, in porcine and bovine muscles differing in rate of contraction and in oxidative capacity. The activity of GDE, the activity of phosphorylase, total glucose content, lactate content and pH were measured from meat samples taken 35 min post-mortem and ultimate pH 24 or 48 h post-mortem.
Both GDE and phosphorylase are needed for the complete degradation of glycogen. In porcine muscles the activities of these glycogen degrading enzymes were higher than in bovine muscles. The activities were increasing with the increasing fast twitch and glycolytic character of a muscle of a given species. However, the increase in the activity of phosphorylase was greater than the increase in the activity of GDE. It was concluded that the GDE may restrict the rate of glycolysis in fast twitch muscles.

Lactic acid bacteria (LAB) isolated from marinated or non-marinated, modified atmosphere packaged (MAP) broiler leg products and air samples of a large-scale broiler meat processing plant were identified and analyzed for their phenotypic properties. Previously, these strains had been found to be coccal LAB. However, the use of a 16 and 23S rRNA gene RFLP database had not resulted in species identification because none of the typically meat-associated LAB type strains had clustered together with these strains in the numerical analysis of the RFLP patterns. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA–DNA hybridization experiments were done. The 16S rRNA gene sequences of three isolates possessed the highest similarities (over 99%) with the sequence of S. parauberis type strain. However, in the numerical analysis of HindIII ribopatterns, the type strain did not cluster together with these isolates. Reassociation values between S. parauberis type or reference strain and the strains studied varied from 82 to 97%, confirming that these strains belong to S. parauberis. Unexpectedly, most of the broiler meat-originating strains studied for their phenotypical properties did not utilize lactose at all and the same strains fermented also galactose very weakly, properties considered atypical for S. parauberis. This is, to our knowledge, the first report of lactose negative S. parauberis strains and also the first report associating S. parauberis with broiler slaughter and meat products.

Yersinia enterocolitica 4/O : 3 is the most frequent cause of sporadic human yersiniosis in Finland and Germany. To investigate the possible link between pigs and humans, 282 human and 534 porcine strains from Finland and Germany were characterized with PFGE using NotI, ApaI and XhoI enzymes. Most of the human strains (>80 %) were indistinguishable from the porcine strains in both countries and most of the genotypes (178/182) were different in Finland and Germany. The indistinguishable genotypes among human and porcine strains together with different genotypes in Finland and Germany indicate that pigs are an important source of sporadic yersiniosis in both countries.

An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species.

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum (C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.

Clostridium botulinum type B was detected by multiplex PCR in the intestinal contents of a suddenly deceased 11-week-old infant and in vacuum cleaner dust from the patient's household. C. botulinum was also isolated from the deceased infant's intestinal contents and from the household dust. The genetic similarity of the two isolates was demonstrated by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis, thereby confirming that dust may act as a vehicle for infant botulism that results in sudden death.

Microbiological and sensory changes in vacuum-packaged charcoal-broiled river lampreys from three lamprey processing plants were monitored as a function of time at 8°C. The lampreys were examined every 7 days up to 8 weeks for aerobic plate count (APC) and lactic acid bacteria (LAB). The highest mean APC and LAB were 6.01 log CFU/g and 4.86 log CFU/g, respectively. Only 6 out of 15 lots reached an APC value of 7.0 log CFU/g during storage. The sensory scores remained at the baseline levels after 8 weeks´ storage. Twenty-seven isolates were randomly picked from MRS agar and identified to species level using a 16S and 23S rDNA HindIII RFLP (ribotyping) database and sequencing of the 16S rRNA gene if no database match was obtained. Twelve of the 27 isolates were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified by the LAB database. However, they possessed very high (99.9%) 16S gene sequence similarity with either Staphylococcus warneri or Staphylococcus pasteuri type strains. The LAB detected, with the exception of W. halotolerans, have commonly been associated with spoilage of fishery products, but in these vacuum-packaged lampreys they were not the dominant organisms within the developing spoilage population.

A total of 176 lactic acid bacteria (LAB) isolated from a typical Spanish blood sausage called “morcilla de Burgos” were identified by means of phenotypic characteristics and 16S rDNA RFLP (ribotyping). LAB were isolated from “morcilla” of different producers and in different storage periods, that includes unpackaged, vacuum and modified atmosphere packaged “morcilla” and vacuum packed and pasteurised “morcilla”. The knowledge of specific spoilage bacteria of ”morcilla de Burgos” will be useful to design new preservation methods to extent the shelf-life of this product. Identification made according to phenotypic and biochemical characteristics shows the majority of the isolates were heterofermentative LAB (93.2%) and eight different bacterial groups could be distinguished (A-G). W. viridescens was the main species detected (42%). In addition, Leuconostoc spp. (23.9%), W. confusa (11.4%) and Lactobacillus fructosus (5.7%) species were found. Few strains were phenotypically missidentified as Lb. sanfrancisco, Pediococcus spp., Lb. sakei/curvatus and Carnobacterium spp. and 11 strains remained unknown. Most of the leuconostocs were identified as Lc. mesenteroides and Lc. carnosum species. Ribotyping shows a quite good correlation with phenotypic methods, although it has been possible to identify 15 different clusters. W. viridescens and leuconostocs were also the predominant LAB. Strains identified as W. confusa by phenotypic characteristics were resolved in W. confusa and W. cibaria by ribotyping. Neither Carnobacterium piscicola nor Lb. sanfrancisco were identified by means of genotypic method. All Lb. fructosus strains and some more included in different phenotypic groups (17 strains in total) could not be associated with any reference strain (cluster VII). Although some discrepancies exists the combination of phenotypic and genotypic methods led to a better identification and characterization of the strains isolated from “morcilla de Burgos”.

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.